PROJECT SUMMARY Mu-opioid receptors are widely distributed in the central and peripheral nervous system and upon activation have manifold actions including; depression of respiration, activation of the reward pathway, disruption of normal gastrointestinal motility and analgesia. Chronic administration of opioids results in a decline in the response (tolerance) the degree of which differs depending on the agonist and the measure under study. Analgesic tolerance to opioids limits the therapeutic efficacy. The link between the initial activation of receptors and the development of long-term tolerance is the subject of intense study and is dependent on multiple mechanisms. One repeatable and robust measure of opioid action measured on single cells is acute desensitization. This is thought to be an initial adaptive step in the pathway to cellular tolerance. Studies of desensitization have centered on (1) agonist occupancy (2) receptor phosphorylation (3) arrestin binding and (4) receptor internalization. By examining receptors where phosphorylation is blocked this proposal will determine the link between desensitization and the development of long-term tolerance. There are two possible outcomes. One is that the block of desensitization disrupts the development of long-term tolerance measured at the cellular level. The second is that the block of desensitization results in continued signaling that augments homeostatic mechanisms at the cellular and synaptic level that counter the continued activation of receptors. The results from this study will address a long-standing question surrounding the mechanisms that underlie a significant therapeutic problem with the use of opioids as analgesics.